Isolation and characterization of 5T4, a tumour-associated antigen.
The monoclonal antibody (MAb) 5T4 defines a human trophoblast antigen marker with a restricted pattern of expression in normal adult tissues but this antigen is expressed on a variety of carcinomas. The purification of 5T4 antigenic molecules is described from term syncytiotrophoblast by a combination of lectin- and immunoaffinity chromatography and gel filtration giving up to 10,000-fold purification with 70% yield. The antigen is carried by non-associated glycoprotein molecules with an apparent molecular weight of 72 kDa on SDS-PAGE and a neutral pI. Removal of N-linked sugars by N-glycanase reveals a core protein of 42 kDa. Treatment with enzymes that cleave O-linked sugars does not substantially alter the molecular size. The native 5T4 molecules are very resistant to proteolysis until the N-linked sugars are removed or the glycoprotein is denatured and reduced. Glycopeptides generated by these approaches will be suitable for amino acid sequencing.
Hole
N
N
Department of Cancer Biology, Salk Institute, San Diego, CA.
Stern
P L
PL
D015295:Antigens, Tumor-Associated, Carbohydrate / Q000032:analysis / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D008871:Microvilli / Q000276:immunology; D008970:Molecular Weight; D011233:Precipitin Tests / Q000379:methods; D014327:Trophoblasts / Q000276:immunology / Q000648:ultrastructure
D015295:Antigens, Tumor-Associated, Carbohydrate